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. 2018 May 15:6:e4805.
doi: 10.7717/peerj.4805. eCollection 2018.

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study

Bixiao Wang  1 , Chunfang Xi  1   2 , Mingwei Liu  3 , Haichen Sun  4 , Shuang Liu  4 , Lei Song  3 , Hua Kang  1
Affiliations

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study

Bixiao Wang et al. PeerJ. .

Abstract

Background: Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial-mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes.

Methods: A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence.

Results: The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24- BCSCs from MCF-7 cells.

Discussion: This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

Keywords: ALDH1; Breast; Breast cancer; CD44CD24; Cancer stem cell; Fibroblast; Phenotypic transformation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Cell morphology and α-SMA expression cannot be used to distinguish CAFs and NFs, but these fibroblasts differ in their biological functions.
(A and B) CAFs and NFs are both morphologically characterized as large spindle-shaped cells with indented nuclei. Bar: 100 μm. (C and D) Immunohistochemical staining for α-SMA in CAFs and NFs. Both samples are positive for α-SMA expression. Bar: 100 μm. (E and F) Immunohistochemical staining for α-SMA in a cancer tissue and a paracancerous tissue more than 3 cm away from carcinoma. Bar: 100 μm. (G–I) Invasion of MCF-7 cells was affected by CM. Bar: 100 μm.
Figure 2
Figure 2. CAFs and NFs are heterogeneous in terms of their protein expression profiles, cellular functions and related signalling pathways.
(A) Heterogeneity in the expression of selected proteins (fold-change < −2 or >2, P < 0.05) between CAFs and NFs. Blue represents down-regulated expression, and red indicates up-regulated expression. All data are transformed by log2 operation. (B) Gene ontology (GO)-based enrichment analysis of up-regulated proteins in CAFs (orange bars) and in paired NFs (blue bars), including cell component (CC), biological process (BP) and molecular function (MF). (C) KEGG pathway analysis for identifying major biological pathways that were different between CAFs (orange bars) and NFs (blue bars).
Figure 3
Figure 3. Mammosphere formation by MCF-7 cells cultured in CM.
(A) Mammosphere formation in MCF-7 cells in the control, CM-NFs (cultured in CM-NFs) and CM-CAFs (cultured in CM-CAFs) groups. Bar: 100 μm. (B) Number of mammospheres greater than 50 μm in diameter in each group. (C) MFE in each group.
Figure 4
Figure 4. CM from CAFs and NFs can induce phenotypic transition in BCSCs.
(A–C) ALDH1 expression in MCF-7 mammospheres from the three groups based on the ALDEFLUOR assay followed by fluorescence activated cell-sorting (FACS). (D–F) CD24 and CD44 expression in MCF-7 mammospheres from the three groups as detected by FACS. (G and H) Localization of CD44 (green), ALDH1 (red) and DAPI (blue) in mammospheres adhered to glass slides as assessed by immunofluorescence. Bar: 100 μm.
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Grants and funding

This work was supported by the National Natural Science Foundation of China (No. 81172517) and Beijing Breast Prevention Institute of Breast Cancer Prevention and Treatment Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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